Errors in cell division, the process during which replicated DNA is partitioned between two daughter cells, have been linked to diseases and developmental defects. Improper cell division has also been exploited in therapeutic strategies widely used to treat diseases, such as cancer. Accurate chromosome segregation, proper cell cycle progression and repair of DNA damage rely on key proteins recognizing post-translational modifications on histones, which assemble the basic units of chromatin known as nucleosomes. Comprehensively profiling proteins that `read' specific post-translational modifications (or `marks') on histones to regulate chromosome biology has been difficult using conventional approaches. Our goal is to fill this knowledge gap by devising new chemistry-based strategies to profile direct `readers' of histone `marks' in specific cellular contexts. Central to our approach s the conversion of dynamic and weak (typically micromolar) protein-protein interactions into stable associations, via covalent bonds, using photo-cross-linkers. This strategy is combined with state-of-the-art quantitative mass spectrometry to identify `readers' of specific histone `marks'. In the completed project period, we have developed this `chemical proteomics' approach, named CLASPI (cross-linking-assisted and SILAC-based protein identification), and demonstrated its ability to identify new `readers' of histone `marks', including histone H3 methylation and phosphorylation. We will build on our recent publications and preliminary data and will focus on identifying `readers' of two different histone phosphorylation `marks', one that indicates DNA damage and is observed during prolonged mitotic arrest with cytotoxic drugs, and another that associates with chromosomes in dividing cells and depends on the activity of Aurora kinase, a conserved regulator of cell division and a target of anti-cancer drugs. The functional significance of `reading' these histone `marks' at different stages of the cell cycle wil be examined using high-resolution microscopy assays, mouse embryonic fibroblasts with key proteins knocked out, and chemical inhibitors that act on fast time-scales to block the activity of kinases responsible for generating these `marks' in cells. The proposal has three aims: (i) To identify proteins that `read' a phosphorylation `mark' on histone H2AX, (ii) To characterize the functions of histone H2AX phosphorylation-`readers', and (iii) To profile `readers' of histone post-translational modifications in living cells. The proposed research combines chemistry and biology approaches to unravel how histone `marks' are `interpreted' by proteins to ensure stable genome propagation by regulating chromosome segregation, DNA damage repair and cell cycle progression. These studies should also shed light on how chemical inhibitors of cell division kill cancer cells. In addition, the comprehensive profiling of key post-translational modification-dependent protein-protein interactions should lead to the selection of new targets for therapeutic agents. Finally, the approaches we develop are general and can be broadly applied to dissect complex and dynamic networks of protein-protein interactions in cells.